Colonic CX3CR1+ macrophages play a crucial role in the anti-inflammatory effect of LMT503 in DSS-induced colitis
Although the ratio and absolute numbers of colon macrophage populations were not altered, LMT503 treatment resulted in polarization from inflammatory to an anti-inflammatory character. It is known that colon resident macrophages show high expression of CX3CR1. During colitis, Ly6C+ monocyte infiltration to the lamina propria can differentiate into CX3CR1int macrophages which are pro-inflammatory. However, afterwards, further maturation into CX3CR1hi regulatory macrophages drives resolution of inflammation (Bain et al., 2013). These gut CX3CR1hi macrophages can clear out apoptotic neutrophils and support tissue healing (Arandjelovic & Ravichandran, 2015; Castoldi et al., 2023). Thus, CX3CR1+ macrophages are crucial in maintaining gut homeostasis. They play a key role in IBD. To verify that macrophage character shift by LMT503 treatment contributed to DSS-induced colitis alleviation, we used a CX3CR1+ macrophage depletion system. For depletion of CX3CR1+ cells, CX3CR1-DTR mice were i.p. injected with diphtheria toxin during DSS-induced colitis. Under DSS treatment, mice deficient of CX3CR1+ macrophages showed body weight loss and high score of disease activity index, similar to those of WT mice (Figure 6a,b). However, absence of CX3CR1+ macrophages abrogated alleviation of colitis including less body weight loss, low score of disease activity index, and recovered colon length after LMT503 treatment. At day 10, group comparison of body weight changes, disease activity index, and colon length suggested that the effect of LMT503 treatment could be abolished in absence of CX3CR1+ macrophages (Figure 6c-e). After CX3CR1+ macrophage depletion, LMT503 could not control pro-inflammatory cytokines IL-6 (Figure 6f). LMT503 treatment significantly increased IL-10 and IL-22 production in inflamed colon which could be diminished after depletion of CX3CR1+ macrophages (Figure 6g). These data suggest that LMT503 can exert its effect through targeted modulation of CX3CR1+ macrophages to alleviate DSS-induced colitis.